**Methods for Detection and Quantification of Aflatoxins**

Alejandro Espinosa-Calderón, Luis Miguel Contreras-Medina, Rafael Francisco Muñoz-Huerta, Jesús Roberto Millán-Almaraz, Ramón Gerardo Guevara González and Irineo Torres-Pacheco *C.A. Ingeniería de Biosistemas, División de Estudios de Posgrado, Facultad de Ingeniería, Universidad Autónoma de Querétaro, Querétaro, Qro., México* 

#### **1. Introduction**

108 Aflatoxins – Detection, Measurement and Control

Van Egmond H. P., Paulsch W. E. & Schuller P. L. (1978). Confirmatory test for aflatoxin M1

Vargas E. A., Preis R. A., Castro L. & Silva C. M. G. (2001). Co-occurrence of aflatoxins B1,

Waltking A. E. & Wilson D. (2006). Liquid chromatographic analysis of aflatoxin using post-

809-812

*Contaminants*, 18, 11, pp. 981-986

*International*, 89, pp. 678-692, ISSN 1060-3271

on thin layer plate. *Journal of the Association of Official Analytical Chemists*, 61, pp.

B2, G1, G2, zearalenone and fumonisin B 1 in Brazilian corn. *Food Additives and* 

column photochemical derivatization: collaborative study. *Journal of AOAC* 

Mycotoxins are fungal toxic metabolites which naturally contaminate food and feed. aflatoxins (AFs), a kind of mycotoxins, are the main toxic secondary metabolites of some *Aspergillus* moulds such as *Aspergillus flavus*, *Aspergillus parasiticus* and the rare *Aspergillus nomius* (Ali et al., 2005, Alcaide-Molina et al., 2009). Such toxins can be separated into aflatoxins B1, B2, G1, B2a and G2a. Its order of toxicity is B1 > G1 > B2 > G2. Letters 'B' and 'G' refer to its blue and green fluorescence colors produced by these compounds under UV light. Numbers 1 and 2 indicate major and minor compounds, respectively (Weidenbörner, 2001; Hussein & Brasel, 2001). *A. flavus* only produces B aflatoxins, while *A. parasiticus* and *A. nomius* also produce G aflatoxins (Alcaide-Molina et al., 2009).

Aflatoxins are produced on various grains and nuts, e.g., corn, sorghum, cottonseed, peanuts, pistachio nuts, copra, cereals, fruits, oilseeds, dried fruits, cocoa, spices and beer in the field and during storage. AFs occur mainly in hot and humid regions where high temperature and humidity are optimal for moulds growth and toxins production (Ventura et al., 2004; Zollner & Mayer-Helm, 2006). Its presence is enhanced by factors as stress or damage to the crop due to drought before harvest, insect activity, soil type and inadequate storage conditions (Alcaide-Molina et al., 2009).

Aflatoxins, when ingested, inhaled or adsorbed through the skin, have carcinogenic, hepatotoxic, teratogenic and mutagenic effects in human and animals (rats, ferrets, ducks, trout, dogs, turkeys, cattle and pigs) (Anwar-Ul\_Haq & Iqbal, 2004) even at very small concentrations. When aflatoxins B1 is ingested by cows, it is transformed into its hydroxylated product, AFs M1 and M2. Such aflatoxins is secreted in the milk and is relatively stable during milk pasteurization, storage, and preparation of various dairy products (Stroka & Anklam, 2002).

Among the more than 300 known mycotoxins, aflatoxins represent the main threat worldwide. After 1975 there has been an increased concern about the possibility of the presence of carcinogenic mold metabolites, particularly aflatoxins in food and animal feed products. Although aflatoxins are regulated in more than 80 countries, their legislation is not yet completely harmonized at the international level (Cucci et al., 2007). Several

Methods for Detection and Quantification of Aflatoxins 111

the greater will see the effect of aflatoxins marked, the lower the concentration of free

However, the electrodes developed by Tan et al. (2009), were coated with conjugate aflatoxins instead of being coated with specific antibody, whereas the sample was mixed with the antibody. In this manner, some antibodies will be captured by free aflatoxins in the sample and some others by those attached to the electrode. Following that, the electrode is washed and it is placed into a solution with antibodies conjugated with alkaline phosphatase enzyme that binds to the antibodies that are bound to conjugate aflatoxins onto the electrode. After that, the electrode is immersed in the substrate solution in order that

Some methods have been reported the use of simple electrodes (Rameil et al., 2010; Tan et al., 2009), while others have made use of multiple electrodes (Neagu et al., 2009; Piermarini et al., 2007), where the latter has shown to have advantages over the first in that: it is more user friendly; it is possible to carry out many experiments in parallel with different samples;

In order to measure the electrical conductivity in the electrodes there are different techniques, such as intermittent pulse amperometric (IPA), potentiometry, or linear sweep

The intermittent pulse amperometric technique involves the application of a periodic pulse of some duration fixed voltage across the electrodes coated and reference measurement, while the measured current varies depending on the conductivity of the substance. Moreover, in the technique of potentiometry, the measuring electrode coated is immersed in substrate solution without contaminating aflatoxins until a stable electrical potential is obtained, called the potential base. This potential varies depending on the amount of aflatoxins contained in the sample. In the linear sweep voltage technique, the sample is fed

The ability of these techniques to detect aflatoxins depends on many factors, including the type of substrate solution that is used, as is the case reported by Rameil et al. (2010), where it was shown that the use of 3 - (4-hydroxyphenyl ) propionic acid (p-HPPA), being a little toxic substance and does not require the use of organic solvents, can increase the conductivity of the substrate in potentiometry to measure aflatoxins M1 in milk. Another factor is the concentration of antibodies in the lining of the electrode, since the higher concentration of these, it can be reached higher peak current than in IPA technique, although the relationship between antibody concentration and electric current conducted is linear in a certain range, such as Tan et al. (2009) work suggests, where the linear range extends from a dilution of 1:30000 to 1:10000 of antibody against aflatoxins B1 found in rice,

Another point to consider is the detection limit, defined as the maximum decrease in signal equal to three times the standard deviation measured in the absence of aflatoxins to be determined. Detection limits down to 1 pg/ml have been obtained in the measurement of aflatoxins M1 in milk (Neagu et al., 2009); meanwhile, detection of aflatoxins B1 in rice has

There are also other measurement devices, as the case of piezoelectric immunosensors. Piezoelectricity is the property possessed by certain materials in which either generates a potential difference from applied mechanical deformation or vice versa (Webster 1999), so that materials that have this feature can resonate at certain frequencies. One of the most common piezoelectric materials is quartz crystal, used by Jin et al. (2009) as a sensor for

antibodies conjugate react and cause a change in electrical conductivity.

and it reduces the time required for new procedures (Piermarini et al., 2007).

with a voltage which changes linearly, with a fixed slope.

being the latter dilution which gave the best results.

reached the limit 0.06 ng/ml (Tan et al., 2009).

aflatoxins in the sample.

voltage (LSV).

institutions around the world have classified and regulated aflatoxins in food. The European Union (EU) has the most rigorous regulations concerning mycotoxins in food. The limits of AFB1 and total AF in foods are 5 and 10 µg/kg, respectively, in more than 75 countries around the world whilst they are 2 and 4 µg/kg in the European Union (EU) (Herzallah, 2009). The maximum residue levels for total AFs and also for the most toxic of them (AFB1) according to the EU Commission Regulations are 2 and 4 g/kg, respectively. The maximum legal limit for AFM1 in milk is set at 0.05µg/kg (50 ppt) for all EU Member States, and 25 ppt for baby food (Cucci et al., 2007). The European Committee Regulations (ECR) has established the maximum acceptable level of AFB1 in cereals, peanuts and dried fruits for direct human consumption in 4ng/g for total aflatoxins (AFB1, AFG1, AFB2, AFG2) and 2ng/g for AFB1 alone (Ricci et al., 2007). The International Agency for Research on Cancer (IARC) classified aflatoxins as Group 1 of human carcinogens (Alcaide-Molina et al., 2009). In USA, the U.S. Department of Agriculture and the U.S. Food and Drug Administration (FDA) have established an "actionable" level of 15-20 ppb of AFs in animal feed products.

Because of such facts, several methodologies for detection and quantification of AFs have been developed. The principal immunochemical based assay is the widespread enzyme linked immunosorbent assay (ELISA). Other methodologies base their performance upon electrochemical and optical principles such as: chromatography, UV-absorption, spectrometry, fluorescence and immunochemical assay tests. The aforementioned methods require well equipped laboratories, trained personnel, harmful solvents and several hours to complete an assay. Novel methods for detection of aflatoxins try to avoid these disadvantages. Among such novel methods, it can be found: biosensors, electrokinetics, electrochemical transduction, amperometric detection, and adsorptive stripping voltammetry. Each of the aforementioned methodologies has its own advantages and limitations according to sensitivity, easiness of use and cost-effectiveness. The objective of this chapter is to provide a general overview of the different methodologies to detect and quantify aflatoxins in the food analysis field.

### **2. Electrochemicals techniques**

Aflatoxins can be measured by the use of electricity and electrochemical immunosensors. These immunosensors consist of a pair of electrodes (measuring and reference), implemented by using the screen-printing technique. The measuring electrode is coated with specific antibodies which will retain interest aflatoxins in the sample, whereas the other electrode (reference) is commonly made of a combination of Ag / AgCl.

The measurement procedure is similar to that carried out by the ELISA test (Enzyme Linked immunoabsorbent Assay). ELISA process is done by taking a sample of the substance to be measured and mixed with a known portion of conjugated aflatoxins with a special enzyme in a microtiter plate hole, and then it is inserted the measuring electrode. In this way, free aflatoxins in the sample compete for fill the places available (antibodies) in the measuring electrode. After some stabilization time, the measuring electrode is removed from the sample, washed with a buffer solution that removes all traces of the sample and leaves intact the electrode coating with aflatoxins that were captured but are not conjugate. After cleaning procedure, the electrode is introduced in a substrate solution that reacts with enzymes in aflatoxins conjugate, changing the electrical conductivity of the substrate depending on the amount of labeled aflatoxins antibodies attached to the electrode. Thus,

institutions around the world have classified and regulated aflatoxins in food. The European Union (EU) has the most rigorous regulations concerning mycotoxins in food. The limits of AFB1 and total AF in foods are 5 and 10 µg/kg, respectively, in more than 75 countries around the world whilst they are 2 and 4 µg/kg in the European Union (EU) (Herzallah, 2009). The maximum residue levels for total AFs and also for the most toxic of them (AFB1) according to the EU Commission Regulations are 2 and 4 g/kg, respectively. The maximum legal limit for AFM1 in milk is set at 0.05µg/kg (50 ppt) for all EU Member States, and 25 ppt for baby food (Cucci et al., 2007). The European Committee Regulations (ECR) has established the maximum acceptable level of AFB1 in cereals, peanuts and dried fruits for direct human consumption in 4ng/g for total aflatoxins (AFB1, AFG1, AFB2, AFG2) and 2ng/g for AFB1 alone (Ricci et al., 2007). The International Agency for Research on Cancer (IARC) classified aflatoxins as Group 1 of human carcinogens (Alcaide-Molina et al., 2009). In USA, the U.S. Department of Agriculture and the U.S. Food and Drug Administration (FDA) have established an "actionable" level of 15-20 ppb

Because of such facts, several methodologies for detection and quantification of AFs have been developed. The principal immunochemical based assay is the widespread enzyme linked immunosorbent assay (ELISA). Other methodologies base their performance upon electrochemical and optical principles such as: chromatography, UV-absorption, spectrometry, fluorescence and immunochemical assay tests. The aforementioned methods require well equipped laboratories, trained personnel, harmful solvents and several hours to complete an assay. Novel methods for detection of aflatoxins try to avoid these disadvantages. Among such novel methods, it can be found: biosensors, electrokinetics, electrochemical transduction, amperometric detection, and adsorptive stripping voltammetry. Each of the aforementioned methodologies has its own advantages and limitations according to sensitivity, easiness of use and cost-effectiveness. The objective of this chapter is to provide a general overview of the different methodologies to detect and

Aflatoxins can be measured by the use of electricity and electrochemical immunosensors. These immunosensors consist of a pair of electrodes (measuring and reference), implemented by using the screen-printing technique. The measuring electrode is coated with specific antibodies which will retain interest aflatoxins in the sample, whereas the other

The measurement procedure is similar to that carried out by the ELISA test (Enzyme Linked immunoabsorbent Assay). ELISA process is done by taking a sample of the substance to be measured and mixed with a known portion of conjugated aflatoxins with a special enzyme in a microtiter plate hole, and then it is inserted the measuring electrode. In this way, free aflatoxins in the sample compete for fill the places available (antibodies) in the measuring electrode. After some stabilization time, the measuring electrode is removed from the sample, washed with a buffer solution that removes all traces of the sample and leaves intact the electrode coating with aflatoxins that were captured but are not conjugate. After cleaning procedure, the electrode is introduced in a substrate solution that reacts with enzymes in aflatoxins conjugate, changing the electrical conductivity of the substrate depending on the amount of labeled aflatoxins antibodies attached to the electrode. Thus,

electrode (reference) is commonly made of a combination of Ag / AgCl.

of AFs in animal feed products.

quantify aflatoxins in the food analysis field.

**2. Electrochemicals techniques** 

the greater will see the effect of aflatoxins marked, the lower the concentration of free aflatoxins in the sample.

However, the electrodes developed by Tan et al. (2009), were coated with conjugate aflatoxins instead of being coated with specific antibody, whereas the sample was mixed with the antibody. In this manner, some antibodies will be captured by free aflatoxins in the sample and some others by those attached to the electrode. Following that, the electrode is washed and it is placed into a solution with antibodies conjugated with alkaline phosphatase enzyme that binds to the antibodies that are bound to conjugate aflatoxins onto the electrode. After that, the electrode is immersed in the substrate solution in order that antibodies conjugate react and cause a change in electrical conductivity.

Some methods have been reported the use of simple electrodes (Rameil et al., 2010; Tan et al., 2009), while others have made use of multiple electrodes (Neagu et al., 2009; Piermarini et al., 2007), where the latter has shown to have advantages over the first in that: it is more user friendly; it is possible to carry out many experiments in parallel with different samples; and it reduces the time required for new procedures (Piermarini et al., 2007).

In order to measure the electrical conductivity in the electrodes there are different techniques, such as intermittent pulse amperometric (IPA), potentiometry, or linear sweep voltage (LSV).

The intermittent pulse amperometric technique involves the application of a periodic pulse of some duration fixed voltage across the electrodes coated and reference measurement, while the measured current varies depending on the conductivity of the substance. Moreover, in the technique of potentiometry, the measuring electrode coated is immersed in substrate solution without contaminating aflatoxins until a stable electrical potential is obtained, called the potential base. This potential varies depending on the amount of aflatoxins contained in the sample. In the linear sweep voltage technique, the sample is fed with a voltage which changes linearly, with a fixed slope.

The ability of these techniques to detect aflatoxins depends on many factors, including the type of substrate solution that is used, as is the case reported by Rameil et al. (2010), where it was shown that the use of 3 - (4-hydroxyphenyl ) propionic acid (p-HPPA), being a little toxic substance and does not require the use of organic solvents, can increase the conductivity of the substrate in potentiometry to measure aflatoxins M1 in milk. Another factor is the concentration of antibodies in the lining of the electrode, since the higher concentration of these, it can be reached higher peak current than in IPA technique, although the relationship between antibody concentration and electric current conducted is linear in a certain range, such as Tan et al. (2009) work suggests, where the linear range extends from a dilution of 1:30000 to 1:10000 of antibody against aflatoxins B1 found in rice, being the latter dilution which gave the best results.

Another point to consider is the detection limit, defined as the maximum decrease in signal equal to three times the standard deviation measured in the absence of aflatoxins to be determined. Detection limits down to 1 pg/ml have been obtained in the measurement of aflatoxins M1 in milk (Neagu et al., 2009); meanwhile, detection of aflatoxins B1 in rice has reached the limit 0.06 ng/ml (Tan et al., 2009).

There are also other measurement devices, as the case of piezoelectric immunosensors. Piezoelectricity is the property possessed by certain materials in which either generates a potential difference from applied mechanical deformation or vice versa (Webster 1999), so that materials that have this feature can resonate at certain frequencies. One of the most common piezoelectric materials is quartz crystal, used by Jin et al. (2009) as a sensor for

Methods for Detection and Quantification of Aflatoxins 113

analyze agricultural products and plants. It has advantages as: simplicity of operation; availability of many sensitive and selective reagents for detection and confirmation without interference of the mobile phase; ability to repeat detection and quantification; and cost effectiveness analysis, because many samples can be analyzed on a single plate with low solvent usage, and the time that TLC employs to analyze the sample is less that LC method (Sherma, 2000; Fuch et al., 2010). The most important differences between TLC and HPTLC are: the different particular size of stationary phase; the care used to apply the samples; and

Diprossimo et al. (1996) present a work where show that TLC was superior to the methods of BF (Best food) CB-RCS-Mod (modified CB method-Rapid Modification of the Cottonseed Method) in terms of less fluorescence interferences, better solvent efficiency, and lower detection levels. Results obtained using TLC method compared to HPLC and enzyme-linked immunosorbent assay (ELISA) was found to agree among method but TLC was least

Papers that use TLC methods to detect and quantify aflatoxins use sample clean-up based on immunoaffinity columns. Therefore, they avoid interfering compounds and allow visual quantification of aflatoxins at concentrations of less than 1 ng/g (Stroka et al., 2000). Immnunoaffinity procedures provide very clean extracts because the sample is cleaned of interference substances. It also permits an easy aflatoxins determination, since they are applicable for automated sample clean-up (Stroka et al., 2002). Because of the advantages of this method, researches have been focused on them to develop new techniques to improve

As aforementioned, HPLC is one of the most common methods to detect and quantify aflatoxins in food. It has been used jointly with techniques such as UV absorption, fluorescence, mass spectrometry and amperometric detectors. Elizalde-González et al. (1998) analyzed aflatoxins B1,B2, G1 and G2 based on HPLC and amperometric detection, and report that it is possible to detect 5 ng of all four aflatoxins. This proposed method is recommended for detection and quantification of the less toxic aflatoxin B2, which is presented in grains. Quinto et al. (2009) proposed a new method for determine aflatoxins B1, B2, G1, and G2 in cereal foods. This method is based on solid phase microextraction coupled with HPLC and a post-column photochemical derivatization to improve the fluorescence of analytes and fluorescence detection. Such method is fast compared with the complete analytical process that uses Immunoaffinity column. However, its sensibility is below the legal limits. Vosugh et al. (2009) present a work that uses HPLC in conjunction with diode array detector (DAD) and a second order iterative algorithm called parallel factor analysis (PARAFAC). Such method is used for quantifing aflatoxins B1, B2, G1, and G2 in pistachio nuts, this work also use a solid phase extraction stage as a clean-up procedure. Manneta et al. (2005) presents a new method with fluorescence detection using pyridinium hydrobromide perbromide as a post-column derivatization agent to determine aflatoxin M1 in milk and cheese. The detection limits obtained were of 1 ng/kg for milk and 5 ng/kg for cheese that are 50-fold lower than the maximum residue level (MRL) for AFM1 in milk and

the way to process the obtained data (Fuch et al., 2010).

the methodologies for quantification of aflatoxins.

**3.3 High Performance Liquid Chromatography (HPLC)** 

40-fold than MRL for AFM1 in cheese set by various European countries.

An interesting application of HPLC is the combination of immobilized enzyme reactor (IMER) in on-line high performance liquid chromatography. This combination allows the selectivity, rapidity and non-destructive, reproducibility of this chromatographic system to

expensive (Schaafsma et al., 1998).

measuring aflatoxins B1 in milk. In this case, the crystal was treated to bind aflatoxins AFB1- BSA conjugate to the material for later subjecting to a similar procedure as mentioned by Tan et al. (2009), differing from this one in that the antibodies attached to conjugate aflatoxins attached to crystal, were marked with gold nanoparticles coated with antibody detector first. The concentration of aflatoxins will be reflected in this case as a change in the resonant frequency of the crystal, as reported by Jin et al. (2009) for the case of aflatoxins B1, where there is a linear relationship between the frequency of resonance and the logarithm of the concentration of aflatoxins.

## **3. Chromatography**

Chromatography is one of the most popular methods to analyze mycotoxins such as aflatoxins. The most common techniques of chromatography are Gas chromatography (GC), liquid chromatography (LC), High performance liquid chromatography (HPLC) and Thinlayer chromatography (TLC). From these methods, LC and HPLC are the most used. In many cases, they are followed by fluorescence detections stage (Cavaliere et al., 2006). LC, TLC and HPLC are the most used quantitative methods in research and routine analysis of aflatoxins (Vosough et al., 2010); these techniques offer excellent sensitivities but they frequently require skilled operators, extensive sample pretreatment and expensive equipment (Sapsford et al., 2006).

#### **3.1 Liquid chromatography**

At the beginning the only separative method was GC, nevertheless, it is restricted to a small set of biological molecules for instance. Those should not be volatiles or should be derivatizated (Roux et al., 2011). LC is other separative method which offers good sensitivity, high dynamic range, versatility and soft ionization conditions that permit access to the molecular mass of intact biological molecules. LC is usually coupled to fluorescence detection stage (FLD), UV absorption and amperometric detection (Elizalde-González, 1998) with pre-column derivatization or post-column derivatization. Extraction and clean up procedures for aflatoxins analysis typically rely on solid phase extraction (SPE) with different absorbent materials. A particular case of SPE is immunoaffinity columns. Improvements have been done, creating techniques based on LC, such as: TLC and Reversed-phase high performance liquid chromatography (RP-HPLC) (Elizalde-González, 1998). LC coupled with fluorescence stage use the aflatoxins fluorescence properties to quantify them. So that, by improve this property it can be obtained better sensibility for aflatoxin detection. The most common techniques to improve fluorescence properties are the use of pre-column derivatization with trifluoretic acid and post-column derivatization with iodine or bromine (Elizalde-González, 1998). Other studies have been done in order to obtain enhancement of the fluorescence emissions of aflatoxins. Franco et al. (1998) collected emission data for AFQ1, AFM1, AFP1 in solvents usually used for their chromatography separation in absence and in presence of different cyclodextrins. Such experiment was made in order to be applied principally in liquid chromatography.

#### **3.2 Thin-Layer Chromatography (TLC)**

Thin-layer chromatography is widely used in laboratories throughout the world for food analysis and quality control. Applications of TLC have been reported in areas of food composition, intentional additives, adulterants, contaminants, etc. TLC has been used to

measuring aflatoxins B1 in milk. In this case, the crystal was treated to bind aflatoxins AFB1- BSA conjugate to the material for later subjecting to a similar procedure as mentioned by Tan et al. (2009), differing from this one in that the antibodies attached to conjugate aflatoxins attached to crystal, were marked with gold nanoparticles coated with antibody detector first. The concentration of aflatoxins will be reflected in this case as a change in the resonant frequency of the crystal, as reported by Jin et al. (2009) for the case of aflatoxins B1, where there is a linear relationship between the frequency of resonance and the logarithm of

Chromatography is one of the most popular methods to analyze mycotoxins such as aflatoxins. The most common techniques of chromatography are Gas chromatography (GC), liquid chromatography (LC), High performance liquid chromatography (HPLC) and Thinlayer chromatography (TLC). From these methods, LC and HPLC are the most used. In many cases, they are followed by fluorescence detections stage (Cavaliere et al., 2006). LC, TLC and HPLC are the most used quantitative methods in research and routine analysis of aflatoxins (Vosough et al., 2010); these techniques offer excellent sensitivities but they frequently require skilled operators, extensive sample pretreatment and expensive

At the beginning the only separative method was GC, nevertheless, it is restricted to a small set of biological molecules for instance. Those should not be volatiles or should be derivatizated (Roux et al., 2011). LC is other separative method which offers good sensitivity, high dynamic range, versatility and soft ionization conditions that permit access to the molecular mass of intact biological molecules. LC is usually coupled to fluorescence detection stage (FLD), UV absorption and amperometric detection (Elizalde-González, 1998) with pre-column derivatization or post-column derivatization. Extraction and clean up procedures for aflatoxins analysis typically rely on solid phase extraction (SPE) with different absorbent materials. A particular case of SPE is immunoaffinity columns. Improvements have been done, creating techniques based on LC, such as: TLC and Reversed-phase high performance liquid chromatography (RP-HPLC) (Elizalde-González, 1998). LC coupled with fluorescence stage use the aflatoxins fluorescence properties to quantify them. So that, by improve this property it can be obtained better sensibility for aflatoxin detection. The most common techniques to improve fluorescence properties are the use of pre-column derivatization with trifluoretic acid and post-column derivatization with iodine or bromine (Elizalde-González, 1998). Other studies have been done in order to obtain enhancement of the fluorescence emissions of aflatoxins. Franco et al. (1998) collected emission data for AFQ1, AFM1, AFP1 in solvents usually used for their chromatography separation in absence and in presence of different cyclodextrins. Such experiment was made

Thin-layer chromatography is widely used in laboratories throughout the world for food analysis and quality control. Applications of TLC have been reported in areas of food composition, intentional additives, adulterants, contaminants, etc. TLC has been used to

the concentration of aflatoxins.

equipment (Sapsford et al., 2006).

in order to be applied principally in liquid chromatography.

**3.2 Thin-Layer Chromatography (TLC)** 

**3.1 Liquid chromatography** 

**3. Chromatography** 

analyze agricultural products and plants. It has advantages as: simplicity of operation; availability of many sensitive and selective reagents for detection and confirmation without interference of the mobile phase; ability to repeat detection and quantification; and cost effectiveness analysis, because many samples can be analyzed on a single plate with low solvent usage, and the time that TLC employs to analyze the sample is less that LC method (Sherma, 2000; Fuch et al., 2010). The most important differences between TLC and HPTLC are: the different particular size of stationary phase; the care used to apply the samples; and the way to process the obtained data (Fuch et al., 2010).

Diprossimo et al. (1996) present a work where show that TLC was superior to the methods of BF (Best food) CB-RCS-Mod (modified CB method-Rapid Modification of the Cottonseed Method) in terms of less fluorescence interferences, better solvent efficiency, and lower detection levels. Results obtained using TLC method compared to HPLC and enzyme-linked immunosorbent assay (ELISA) was found to agree among method but TLC was least expensive (Schaafsma et al., 1998).

Papers that use TLC methods to detect and quantify aflatoxins use sample clean-up based on immunoaffinity columns. Therefore, they avoid interfering compounds and allow visual quantification of aflatoxins at concentrations of less than 1 ng/g (Stroka et al., 2000). Immnunoaffinity procedures provide very clean extracts because the sample is cleaned of interference substances. It also permits an easy aflatoxins determination, since they are applicable for automated sample clean-up (Stroka et al., 2002). Because of the advantages of this method, researches have been focused on them to develop new techniques to improve the methodologies for quantification of aflatoxins.

#### **3.3 High Performance Liquid Chromatography (HPLC)**

As aforementioned, HPLC is one of the most common methods to detect and quantify aflatoxins in food. It has been used jointly with techniques such as UV absorption, fluorescence, mass spectrometry and amperometric detectors. Elizalde-González et al. (1998) analyzed aflatoxins B1,B2, G1 and G2 based on HPLC and amperometric detection, and report that it is possible to detect 5 ng of all four aflatoxins. This proposed method is recommended for detection and quantification of the less toxic aflatoxin B2, which is presented in grains. Quinto et al. (2009) proposed a new method for determine aflatoxins B1, B2, G1, and G2 in cereal foods. This method is based on solid phase microextraction coupled with HPLC and a post-column photochemical derivatization to improve the fluorescence of analytes and fluorescence detection. Such method is fast compared with the complete analytical process that uses Immunoaffinity column. However, its sensibility is below the legal limits. Vosugh et al. (2009) present a work that uses HPLC in conjunction with diode array detector (DAD) and a second order iterative algorithm called parallel factor analysis (PARAFAC). Such method is used for quantifing aflatoxins B1, B2, G1, and G2 in pistachio nuts, this work also use a solid phase extraction stage as a clean-up procedure. Manneta et al. (2005) presents a new method with fluorescence detection using pyridinium hydrobromide perbromide as a post-column derivatization agent to determine aflatoxin M1 in milk and cheese. The detection limits obtained were of 1 ng/kg for milk and 5 ng/kg for cheese that are 50-fold lower than the maximum residue level (MRL) for AFM1 in milk and 40-fold than MRL for AFM1 in cheese set by various European countries.

An interesting application of HPLC is the combination of immobilized enzyme reactor (IMER) in on-line high performance liquid chromatography. This combination allows the selectivity, rapidity and non-destructive, reproducibility of this chromatographic system to

Methods for Detection and Quantification of Aflatoxins 115

When small amounts of organic solvents are used in the buffer system good separation of aflatoxins are achieved. Nonetheless, it has been probed only with standard buffers (Gilbert

All the aflatoxins have a maximum absorption around 360 nm (Akbas and Ozdemir, 2006). Letters 'B' and 'G' of the aflatoxins refer to its blue (425nm) and green-blue (450nm) fluorescence colours produced by these compounds under Ultra Violet (UV) light. AFB1 is the most common aflatoxin; it is followed by the AFB2. AFG is fairly rare. The fluorescence emission of the G toxin is more than 10 times greater than that for the B toxin (Alcaide-

The black light test is a method which correctly identifies negative AFs samples with minimum expenditure of time and money. It consists on the illumination of the sample with a UV lamp. Tests should be made in a darkened area for best contrast. Fluorescence may be bright or dim, depending on the amount of fluorescing agent present. Polished metal surfaces reflect blue light, thus, users must be careful distinguishing fluorescence from such reflection. It is highly recommended to use safety goggles when working with the black light test. These goggles eliminate blue haze resulting from eye fluorescence caused by

However, fluorescence does not happen exclusively when aflatoxins are present. There are other substances in food that fluoresce under long wave UV radiation. Fungi as *Aspergillus niger*, various *Penicillium* species, *Aspergillus repens* and other species do not produce aflatoxins, but may produce fluorescent harmless metabolites. Then, it can be said that fluorescence is not a specific indication of the presence of aflatoxins, although it may indicate that conditions have been favourable for growth of toxic molds (B-100 Series

Furthermore, fluorescence is not stable. It disappears in 4 to 6 weeks of continuous exposure to visible or UV radiation although the toxin remains. Therefore, fresh samples must be taken. Hence, the reliability of the method depends on the size of the sample taken for analysis and how it is taken. A sample must be large enough to be representative of the entire lot and must be taken from all parts of the lot (B-100 Series

The black light test is commonly applied on animal feed. However, it is only a preliminary confirmatory test; it does not give a quantitative indication. Thus confirmatory and quantitative measurements are needed to be applied to those samples that reacted positively to the black light test. Non-fluorescing samples need not be subjected to this. A quantitative screening test which commonly follows the black light test is small chromatographic column (mini-column) (B-100 Series Ultraviolet Lamps, UVP). After the quantitative test a judgment

LIF detection technique was pioneered by Yeung (Novotny & Ishii, 1985). This screening method consists on a mobile phase which contains an eluted sample of aflatoxins. Such

Different techniques for detection of AFs related to fluorescence are exposed bellow.

& Vargas, 2003).

**4. Fluorescence** 

Molina et al., 2009).

**4.1 Black light test** 

reflected longwave UV radiation.

Ultraviolet Lamps, UVP).

Ultraviolet Lamps, UVP).

can be made as to whether or not accept a lot.

**4.2 Laser-Induced Fluorescence (LIF) screening method** 

be combined with the specification and sensitivity for an enzymatic reaction (Girelli & Mattei, 2005). Derivatization with a fluorophore enhances the natural fluorescence of aflatoxins and improves detectability. The pre-column approach uses the formation of the corresponding hemiacetals using trifluoroacetic acid (TFA), while the post-column one utilizes either bromination by an electrochemical cellor in addition of bromide, or pyridinium hydrobromide perbromide, for the mobile phase and the formation of an iodine derivative.

Even though the optical devices have dominated the traditional methods for HPLC, the present trend is to use mass detectors in the different HPLC types and configurations. This is because of the universal, selective and sensitive detection they provide (Alcaide-Molina, 2009).

There are several techniques that use chromatography for aflatoxin analysis in food (principally in milk, cheese, corn, peanuts, nuts). Commonly the quantification of the aflatoxins is made by a fluorescence detector that takes advantage of fluorescence properties of aflatoxins under determined wavelength. As a result, researchers have been focused on improving these fluorescence properties to develop more sensitive methods than the commonly used so far. Currently techniques such as pre-column derivatization and postcolumn derivatization are commonly used to improve aflatoxins fluorescence properties. They also have a clean-up stage to obtain a more pure sample, permiting a better quantification. Some of the common methods used in the clean-up stage are: immunoaffinity column and solid phase extraction.

#### **3.4 Electrokinetics**

HPLC is a method for detection of aflatoxins which often is enhanced by other techniques, resulting on alternative chromatographic methods. Accomplishing techniques related to electrokinetics are: Micellar electrokinetic chromatography (MEKC), reversed flow micellar electrokinetic chromatography (RFMEKC), and capillary electrokinetic chromatography (CEKC) with multiphoton excited fluorescence (MPE) detection, among others (Gilbert & Vargas, 2003).

Electrokinetics consists on an interfacial double layer of charges effect in heterogeneous fluids (Rathore and Guttman, 2003). Such effect generates the motion of the fluid due to an external force. This external force may be of different natures, but it is called electrophoresis when the force is an electric field; and capillary osmosis when the force is a chemical potential gradient and the motion of liquid happens in a porous body.

Capillary electrophoresis is a technique that although not been widely available as an alternative in many laboratories which routinely conduct HPLC, it has the advantage that it avoids the use of organic solvents. aflatoxin B1 can be determined by capillary electrophoresis (CEKC) with laser-induced fluorescence (LIF) detection (Maragos & Greer, 1997) after a clean-up process comparable to that required for HPLC, and with a very similar sensitivity to it. Besides, Electrophoresis does not require derivatization of aflatoxins, being that an advantage over HPLC. Sensitivity on CEKC can be further improved by using multiphoton excitation. Detection at levels 104 better than previously achieved by capillary separation in less than 90 seconds can be reached, which demonstrates the potential of this technique (Wei et al., 2000).

Micellar electrokinetic chromatography (MEKC) is conducted in polyacrylamide-coated capillaries under almost complete suppression of electroosmotic flow (Janini et al., 1996). When small amounts of organic solvents are used in the buffer system good separation of aflatoxins are achieved. Nonetheless, it has been probed only with standard buffers (Gilbert & Vargas, 2003).
